Biotherapy for viral infections using biopolymer based micro/nanogels

ABSTRACT

A method of treatment for HIV or other viral infection involves the administration of a biopolymer based hydrogel nanoparticles and/or microparticles that are comprised of chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol. These biopolymer based hydrogel nanoparticles and/or microparticles are the antiviral agent that can be employed alone or in combination with other drugs for treatment of the viral infection. Evidence demonstrates the pre-treatment with nanogels is highly effective at lowering HIV growth. Therefore, this antiviral biopolymer based hydrogel nanoparticles and/or microparticles may also be employed as a prophylactic.

This invention was made with government support under DA040537 and DA037838, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF INVENTION

Hydrogels are soft materials developed using natural and synthetic polymers that have been explored intensively in biomedical applications. Hydrogels are the three-dimensional network of hydrophilic polymers which have the ability to imbibe large amount of water. Their soft porous structure makes them resemble human tissue, which serves as prefect candidate to be used in drug delivery and tissue engineering applications. Hydrogels can be designed and developed in various forms such as films, crystals and particles for drug delivery application. The development of natural polymer based hydrogels for the treatment of various infectious diseases is of great interest in the field of infectious diseases. Hydrogels with microgel and nanogel dimensions show improved prospective in imaging, therapeutics delivery and tissue engineering.

According to a 2017 UNAIDS report, 36.9 million people worldwide are currently affected with HIV/AIDS, which includes 1.8 million children. Challenged by the growing threats of highly active antiretroviral therapy (HAART) that involve two or more drugs used in combination, nanomedicines have been explored for the development of biomaterials having inherent therapeutic efficacy and that can be used for diagnostics. Micro/nanogels are promising as the next generation materials for therapeutics. Biopolymers, such as chitosan and hydroxyethyl cellulose, have been explored in formulations that display anti-viral and anti-bacterial properties. Sulfated chitosan derivatives have been prepared that inhibit retrovirus replication. N-carboxymethylchitosan-N,O-sulfate inhibits the synthesis of virus-specific proteins and replication of HIV-1 in cultured T-cells and Rausher murine leukemia virus; displaying no cytotoxicity of chitosan derivative toward the cell cultures. (see Chirkov, Prikladnaya Biokhimiya i Mikrobiologiya 2002, 38, 1, 5-13) Hydroxyethyl cellulose gels have been used as a carrier of chloroquine that is stable at ambient tropical conditions for treatment of HIV-1 infection. (see Brouwers et al., Virology 2008, 378, 306-10)

Recently, auto-fluorescent hydrogel nanoparticles and microparticles based on chitosan and hydroxyethyl cellulose using linseed oil polyol and a crosslinking agent have been designed (U.S. patent application Ser. No. 15/907,703). The overall objective is to use these auto-fluorescent hydrogel nanoparticles and microparticles in the treatment of HIV and other viral agents.

BRIEF SUMMARY

Embodiments of the invention are directed to a method of treatment for a viral infection where treatment involves administering a biopolymer based hydrogel nanoparticles and/or microparticles. The biopolymer based hydrogel nanoparticles and/or microparticles comprise chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol. Administrating can involve applying an aqueous suspension comprising the biopolymer based hydrogel nanoparticles and/or microparticles. The aqueous suspension can be within a water-in-oil emulsion or an oil-in-water emulsion. The aqueous suspension can be applied to a surface prior to contact with a virus-containing fluid or virus, essentially the biopolymer based hydrogel nanoparticles and/or microparticles can also be used as a prophylactic. Alternatively, or additionally, the body surface can be treated after contact with a fluid suspected to contain a virus. The aqueous suspension can also contain a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof. The method of administrating can be by ingesting a formulation comprising the biopolymer based hydrogel nanoparticles and/or microparticles. The formulation can be in the form of a solid device such as a powder, pill, or capsule. The solid formulation can include a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof. The method of administrating can be by injecting an aqueous suspension containing the biopolymer based hydrogel nanoparticles and/or microparticles. The injectable suspension can include a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof. The viral infection can be an HIV infection.

An embodiment of the invention is directed to a formulation for treatment or prevention of an HIV infection, where a biopolymer based hydrogel nanoparticles and/or microparticles comprising chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol is advantages for treatment of a viral infection. The formulation can be based on an aqueous suspension comprising the biopolymer based hydrogel nanoparticles and/or microparticles or can be in the form of a solid powder, pill, or capsule. The formulation can include a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof. The formulation can also include any adjuvant to promote administration of the formulation or augment the formulation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows a bar graph for HIV infection as measured by p24 ELISA values 2 hours post-HIV exposure for PHA-stimulated peripheral blood mononuclear cells (PBMCs) (50×10⁶) infected with HIV(NLAD8 strain) with the plates pretreated for 24 hour pretreatment with nanogel, according to an embodiment of the invention, where culture supernatants were collected 1, 4, 6, 8, and 10 days post infection (dpi).

FIG. 1B shows a bar graph for HIV infection as measured by p24 ELISA values 2 hours post-nanogel addition to HIV infection for PHA-stimulated PBMCs (50×10⁶) infected with HIV(NLAD8 strain) 2 hours prior to addition of the nanogels which was 2 hours prior to analysis, according to an embodiment of the invention, where culture supernatants were collected 1, 4, 6, 8, and 10 dpi.

FIG. 1C shows a bar graph for HIV infection as measured by p24 ELISA values 10 dpi for nanogel pre-treated and post-treated with nanogels according to embodiments of the invention.

FIG. 2A shows a bar graph for HIV p24 ELISA values for HA infected with HIV(NLAD8 strain) 16 hours post infection for increasing doses of nanogel added to the cell culture with supernatant collected 7 days post infection(dpi).

FIG. 2B shows a bar graph of quantified Long terminal repeat(LTR) transcripts via digital droplet polymerase chain reaction (ddPCR) for HIV-infected HA harvested 7 dpi with total RNA isolated with statistical significance determined by ANOVA,** p<0.01 and *p<0.05 Post hoc analysis Dunn's test.

FIG. 3 shows a transmission electron microscopy (TEM) image for the hydrogel nanoparticles.

FIG. 4A shows a bar graph of cytocompatibility testing for nanogels at various concentrations (5-100 μg) as a function of time for HAs.

FIG. 4B shows a bar graph of cytocompatability testing for nanogels at various concentrations (5-100 μg) as a function of time for PBMCs.

FIG. 4C shows a bar graph of cytocompatibility testing for nanogels at various concentrations (5-100 μg) as a function of time for CHME-5.

DETAILED DISCLOSURE

In an embodiment of the invention, biopolymer based hydrogel nanoparticles and microparticles of chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol have been discovered to display inherent anti-HIV properties. Auto-fluorescent hydrogels in micro and nano scales from completely natural polymers chitosan, HEC and sustainable resource linseed oil based polyol exhibit complete biocompatibility over a concentration range of 10-100 μg using a wide range of host cells, such as, astrocytes, peripheral blood mononuclear cells (PBMCs) and microglia. These hydrogels display a dynamic wide range of emission wavelengths, 450 to 750 nm and 710 to 810 nm, which permits simultaneous in vivo imaging. Their high stability in aqueous solution at physiological pH, 7.4, allows good shelf-life in solution and in a dry form at room temperature, for at least 6 months, while retaining their auto-fluorescence property. The use of a wide range of sizes, from microscale to nanoscale, results in cellular uptake and co-localization in ex-vivo studies with PBMC, microglia and astrocytes. Remarkably, the hydrogel particles transmigrate the blood-brain barrier, which allows their use for the drug delivery to the central nervous system.

The micro/nanogels according to an embodiment of the invention, display anti-viral properties against HIV primary cellular targets, PBMCs and human astrocytes (HAs), during early and late stages of the HIV life cycle. Level of expression of Capsid antigen (p24) of newly released virions representing late stage in HIV replication was determined using p24 enzyme-linked immunosorbent assay (ELISA) and early stage HIV transcription was measured by quantifying the number of gene transcripts containing the HIV long terminal repeat (LTR) using digital droplet PCR (ddPCR).

Nanogel anti-viral properties were assessed in HIV primary cellular targets, PBMCs and Human astrocytes, during early and late stages in the HIV life cycle. Level of expression of Capsid antigen (p24) of newly released virions representing late stage in HIV replication was determined using p24 ELISA while early stage HIV transcription was measured by quantifying the number of gene transcripts containing the HIV LTR using digital droplet PCR (ddPCR).

PBMCs were infected with HIV-1 pre- or post-nanogel administration. Culture supernatants were collected over 10-day period and p24 ELISA performed to measure viral replication. Preliminary data shows that pretreatment with increasing doses of T4 nanogel significantly lowered p24 detection levels with little to no p24 released over the 10 day infection period, as shown in FIG. 1A. It appears that nanogel treatment post HIV exposure allowed low-level HIV replication, but was also able to significantly reduce p24 levels in HIV-infected PBMCs, as shown in FIG. 1B. Pre-exposure to nanogel diminished p24 levels compared to post exposure, as indicated in FIG. 1C, which indicates that nanogel is a novel pre-exposure HIV prophylactic.

HIV infected HA treated with nanogel post HIV exposure exhibited 10-fold and 5-fold reduction in p24 and LTR transcripts, respectively, as shown in FIG. 2A and FIG. 2B, respectively. This evidence indicates that nanogel significantly disrupts the HIV life cycle such that both viral transcription and release are greatly reduced.

The biopolymer based hydrogel nanoparticles and microparticles of chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol can be used in conjunction with any vehicle for administration. The nanoparticles or microparticles can be used in a solid formulation as a pill or a capsule. The nanoparticles or microparticles can be used as an aqueous suspension formulation to introduce to a patient by an injection, for the topical application to an internal or external body surface, or within a liquid filled capsule. The aqueous solution can be employed in a water-in-oil emulsion wherein the continuous phase vehicle of the oil can be exploited for surface properties to retain the nanoparticle and/or microparticle formulation in a selected environment of the body. The aqueous solution can be employed in an oil-in-water emulsion wherein the continuous phase vehicle of the aqueous suspension can be augmented by oil-soluble or oil-suspendable adjuvants. The formulation can include adjuvants that include, but are not exclusive to one or more of: nutrients, surfactants, thickeners, other viscosity modifiers, fillers, and other drugs. The drugs can be other antiviral agents, antibacterial agents, antifungal agents, or any combination of one or more of these drugs.

The biopolymer based hydrogel nanoparticles and/or microparticles comprising formulation can be used as a prophylactic agent, such that it can be used prior to potential exposure to HIV or other viruses. For example, a suspension of the nanoparticles or microparticles can be contacted with surfaces of the genitals or other surfaces contacted during intimate contact between persons. Pills or capsules can also be ingested prior to intimate contact in a prophylactic capacity.

Alternately, the biopolymer based hydrogel nanoparticles and/or microparticles comprising formulation can be used by a patient after contracting HIV or other viral infection. The formulations can be used in regiments that employ other agents for compound therapies where the nanoparticle and/or microparticles are used simultaneously or non-simultaneous administration to attack the virus or relieve other symptoms or combat secondary infections with other virus, bacteria, or fungi to the compromised patient.

Methods and Materials

Chitosan (448877-50G, Sigma Aldrich), HEC, Heavy liquid paraffin oil (0.8660-0.890 g/cm³), Tween 80, Ethanol, n-Hexane (Sigma Aldrich), Glycine (75.07 g/mol, 1.607 g/cm³), linseed oil, glacial acetic acids, hydrogen peroxide, diethylether, and acetic anhydride (Sigma Aldrich) were used as received. Linseed oil polyol was prepared using the method of Sharmin et al., International Journal of Biological Macromolecules, 2007, 40, 407-22. Deionised water from a Millipore mille U10 water purification system was used in the preparation of hydrogels and other In vitro experiments.

Preparation of Micro/Nanogels

Hydrogel nanoparticles of chitosan (0.7 g) and HEC (0.3 g) with linseed oil based polyol as a hydrophobic modifier 1 ml of 2%), were prepared in a beaker using a water-in-oil emulsion polymerization method. Polymer solution (20 ml of 2% (w/v)) was prepared in 1% (v/v) acetic acid. A separate beaker was used to make a mixture of liquid paraffin oil and 1% (w/w) Tween 80. The polymer solution was added dropwise to the mixture of oil and surfactant stirred at 14000 rpm on a magnetic stirrer. The mixing of the solution was continued for 20 minutes followed by the addition of glutaraldehyde (5 ml) for another 10 minutes. The linseed oil polyol was added to the reaction mixture and stirring was continued at 14000 rpm for 5 hours. The particles were washed thoroughly with n-hexane to remove excess oil. Any excess glutaraldehyde was deactivated by 0.1 M glycine. The washed hydrogel particles were dried at room temperature. A TEM image of the nanoparticles is shown in FIG. 3.

Characterization of Micro/Nanogels Using FT-IR, Raman and TEM Analysis

The hydrogel samples were dried under vacuum overnight till attaining a constant weight. The dried samples were analyzed using model 1750 FT-IR spectrophotometer (PerkinElmer Cetus Instruments, Norwalk, Conn.). TEM analysis was performed using Phillips CM-200 200 kV transmission electron microscope with an operating voltage of 80 kV.

Isolation and Culture of PBMC, Microglial (CHME5) and HA

PBMCs isolation was carried out using leukopacks (buffy coat), obtained commercially from a community blood bank (One Blood, Miami, Fla., USA). PBMCs were isolated using a common protocol. (see Atluri et al. Scientific Reports, 2016, 6, 27864) The buffy coat is diluted with phosphate buffer saline (PBS) (Invitrogen, Gaithersburg, Md.) at room temperature. The diluted whole blood was overlaid on the top of a Ficoll-Histopaque centrifuge such that three separate layers—plasma, buffy coat, and red blood cells (RBCs) were formed. The samples were centrifuged at 1,200 g for about 40 min at room temperature. The buffy coat layer formed at the interface of plasma and red blood cells. The buffy coat containing PBMCs collected and the cells were washed with PBS. The pellet was re-suspended in Ammonium-Chloride-Potassium (ACK) lysing buffer in order to lyse the RBCs in the samples. The suspension was kept in ice for 15 minutes. Cells were washed a second time with PBS. The total cell number and cell viability was evaluated by trypan blue (Sigma, St. Louis, Mo.) exclusion. The PBMC pellet were re-suspended in a culture medium of Roswell Park Memorial Institute (RPMI) 1640 (Life Technologies, Gaithersburg, Md.), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma, St. Louis, Mo.), 2 mM glutamine (Sigma, St. Louis, Mo.), 100 μs streptomycin (Sigma, St. Louis, Mo.), 100 U penicillin (Sigma, St. Louis, Mo.), and 10% fetal bovine serum (Life Technologies, Gaithersburg, Md.).

CHME5, a microglia cell line was cultured using Eagle's minimum essential medium (MEM) supplemented by fetal bovine serum to a final concentration of the antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, Mo.).

Primary HAs were purchased from ScienCell Research laboratories (Carlsbad, Calif.; Cat. #1800-5). These cells were grown on the astrocyte medium purchased from ScienCell laboratories (Cat. #1801) containing 2% of fetal bovine serum (ScienCell Cat. #0010), astrocyte growth supplement (ScienCell Cat. #1852) and penicillin/streptomycin (ScienCell Cat. #0503), antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, Mo.).

Biocompatibility Assessment

Biocompatibility was assessed for HAs, PBMCs and CHME5 using a XTT cell viability assay with sodium 3,3′+[(phenlamino)carbonyl]-3,4-tetrazolium)-bis(4-methoxyl-6-nitro)benzene sulfonic acid hydrate, as shown in FIGS. 4A-4C, respectively. Primary HAs (1×104 cells per well) were seeded in a 24 well plate and after 24 h of incubation at 37° C., the medium was replaced with 1 ml of fresh medium containing nanogel 5-100 ug/ml. Cells were treated with various concentrations and incubated for 1, 2, 4, and 7 days. XTT, 1 mg/ml, and 2.5 μl of freshly prepared phenazinemethosulfate (PMS) solution were added to each well.

The XTT containing wells were incubated for 4 h at 37° C. A multi-mode microplate reader (Synergy HT), was used to measure absorbance at 450 nm wavelength. All experiments were performed in triplicate. Statistical analysis was performed as a one way analysis of variance (ANOVA) and using Tukey's multiple comparison test and difference considered was P<0.05.

CHME5 and PBMCs, 2×105 cells per well were seeded in 24 well plate. The protocol described above for HAs was followed for each cell type. To maintain the PBMCs for 7 days, fresh media containing IL-2 was added at regular intervals.

Lactate Dehydrogenase (LDH) Cytotoxicity of Nanogel in CHME5 and PBMCs

CHME5 cells and PBMCs (10,000 cells per well) were plated in a 96-well plate and incubated at 37° C., 5% CO₂. After 24 hrs, different concentrations (10 ug-100 ug) of nanogel formulations were added to the culture media and incubated for 24 hrs. The Thermo Scientific Pierce LDH Cytotoxicity Assay Kit was used to quantitatively measure lactate dehydrogenase (LDH) released into the media from damaged cells, which act as a biomarker for the cellular cytotoxicity and cytolysis. For the determination of the LDH background activity, a complete medium control was included. Additional cells were plated in triplicate for spontaneous LDH activity controls (negative control with water) and maximum LDH activity controls (positive control with 10× lysis buffer). Plates were incubated overnight in CO₂ at 37° C. Various concentrations of nanogels were prepared (10 μg to 100 μg) and added to one set of wells. Further, cells were incubated for 24 hours in an incubator at 37° C., 5% CO₂. 10 μL of Lysis Buffer (10×) was added to the wells serving as Maximum LDH Activity Controls and mixed gently by tapping. The plate was further incubated at 37° C., 5% CO₂ for 45 minutes. A 50 μL portion of each sample medium (e.g., complete medium, serum-free medium, Spontaneous LDH Activity Controls, compound-treated and Maximum LDH Activity Controls) was transferred to a 96-well flat-bottom plate in triplicate wells using a multichannel pipette. The plate is incubated at room temperature for 30 minutes in dark.

After 30 min of incubation, 50 μL of stop solution was added to each sample well and mixed gently by tapping. Absorbance was measured at 490 nm and 680 nm. Determination of LDH activity was done by subtracting the 680 nm absorbance value from (background) from the 490 nm absorbance before calculation of % Cytotoxicity.

% Cytotoxicity=Nanogels treated LDH activity-Spontaneous LDH activity/Maximum LDH-Spontaneous-LDH Nanogels treated×100%

Cytotoxicity=Compound-treated LDH activity−Spontaneous LDH activity/Maximum LDH activity−Spontaneous LDH activity×100

Cellular Uptake

CHME5 and PBMCs cells (1×106) were incubated for 24 hours in different concentrations of nanogels (1 to 100 μg). The cells were harvested, washed, and data acquired using Amnis FlowSight and analyzed using Ideas Software. A total of 10,000 events were collected for all samples. Since these nanogels have multichannel fluorescence properties, they were acquired and analyzed through predetermined channel 8 (ex/em: 405 nm/505-560 nm) that demonstrated the maximum intensity.

Statistical Analysis

Test data were represented as Mean±SD of three independent experiments or otherwise indicated. Statistical significance between the two groups determined using Student t-Test and for multiple experimental groups. One-way ANOVA was used to analyze the significant differences. Samples were considered statistically significantly when p-values equal less than 0.05.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. 

We claim:
 1. A method of treatment for a viral infection, comprising administering a biopolymer based hydrogel nanoparticles and/or microparticles, said biopolymer based hydrogel nanoparticles and/or microparticles comprising chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol.
 2. The method according to claim 1, wherein administrating comprises applying an aqueous suspension comprising said biopolymer based hydrogel nanoparticles and/or microparticles.
 3. The method according to claim 2, wherein the aqueous suspension comprising said biopolymer based hydrogel nanoparticles and/or microparticles is within a water-in-oil emulsion.
 4. The method according to claim 2, wherein applying is to a surface prior to contact with a fluid suspected to comprise a virus, wherein administering said biopolymer based hydrogel nanoparticles and/or microparticles is as a prophylactic.
 5. The method according to claim 2, wherein applying is to a surface after contact with a fluid suspected to comprise a virus, wherein said biopolymer based hydrogel nanoparticles and/or microparticles is a therapeutic.
 6. The method according to claim 2, wherein the aqueous suspension further comprises a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof.
 7. The method according to claim 1, wherein administrating comprises ingesting a formulation comprising said biopolymer based hydrogel nanoparticles and/or microparticles.
 8. The method according to claim 5, wherein the formulation comprises a solid in the form of a powder, pill, or capsule.
 9. The method according to claim 7, wherein the formulation further comprises a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof.
 10. The method according to claim 1, wherein administrating comprises injecting an aqueous suspension comprising said biopolymer based hydrogel nanoparticles and/or microparticles.
 11. The method according to claim 10, wherein the aqueous suspension further comprises a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof.
 12. The method according to claim 1, wherein the viral infection is an HIV infection.
 13. A formulation for treatment or prevention of an HIV infection, comprising a biopolymer based hydrogel nanoparticles and/or microparticles, said biopolymer based hydrogel nanoparticles and/or microparticles comprising chitosan, hydroxyethyl cellulose (HEC), and linseed oil polyol.
 14. The formulation according to claim 13, wherein the formulation is an aqueous suspension comprising said biopolymer based hydrogel nanoparticles and/or microparticles.
 15. The formulation according to claim 14, wherein the aqueous suspension is included in a water-in-oil emulsion or an oil-in-water emulsion.
 16. The formulation according to claim 13, wherein the formulation is a solid powder, pill, or capsule comprising said biopolymer based hydrogel nanoparticles and/or microparticles.
 17. The formulation according to claim 13, further comprising a second antiviral agent, an antibacterial agent, an antifungal agent, or any combination thereof. 